1. Field Of The Invention
This invention concerns a murine monoclonal antibody that recognizes an antigen present on tumors of epithelial origin. This invention also concerns the recognized antigen, in purified form, that reacts with the disclosed antibody. Also of concern are: the cell line that secretes the antibody, radiolabeled antibody, and diagnostic and therapeutic processes that employ the antibody.
2. State Of The Art
Fused cell hybrids of spleen cells and myeloma cells have been described in the literature by Kohler et al. in Nature, Vol. 256, 495 to 497 (1975) and in Eur. J. Immunol. Vol. 6, 511 to 519 (1976); by Milstein et al. in Nature, Vol. 266, 550 to 552 (1977); and by Walsh, Nature, Vol. 266, 495 (1977).
The techinque is also set out in some detail by Herzenberg and Milstein, in Handbook of Experimental Immunology, ed. Weir (Blackwell Scientific, London), 1979, pages 25.1 to 25.7. Relative to the parent myeloma cell line employed herein for the fusion event, see Kearney et al. J. Immunol., 123, 1548 to 1550 (1979).
Patents relating to antibodies against human tumors produced by hybrodoma technology include U.S. Pat. Nos. 4,172,124 and 4,196,265. Representative of the art concerning monoclonal antibodies that have specificity for antigens on carcinoma cells are U.S. Pat. No. 4,349,528 and U.S. Pat. No. 4,350,683.
Related publications include the following: Herlyn et al., Proc. Natl. Acad. Sci. USA 76, 1438 to 1442 (1979); Mazauric et al., Canc. Res. 42, 150 to 154 (1982); Canevari et al., Canc. Res. 43, 1301 to 1305 (1983); Colcher et al., Proc. Natl. Acad. Sci. USA 78, 3199 to 3203 (1981); Ueda et al., Proc. Natl. Acad, Sci. USA 78, 5122 to 5126 (1981); Cuttitta et al., Proc. Natl., Acad. Sci. USA 78, 4591 to 4595 (1981); Minna et al., In Vitro 17, 1058 to 1070 (1981). Finally, Menard et al., in Canc. Res. 43, 1295 to 1300 (1983) describe (MBrl) antibodies that react with apocrine sweat glands, not eccrine sweat glands, the antibodies recognizing an antigen which is a lipid.
The immune response to entry of a foreign substance into the body consists of secretion by plasma cells of "antibodies" which are immunoglobulin (Ig) molecules with combining sites that recognize particular determinants on the surface of the foreign substance, or antigen, and bind to them. Usually, the antibody (Ab) response to an antigen (Ag) is heterogeneous. Upon injection of a body with an immunogen, the body manfactures large numbers of antibodies directed against various components of the antigen and even against various determinant sites on the antigen. It is difficult to separate various antibodies and so conventional antisera contain mixtures of antibodies. It has long been a goal to design a source of antibodies that recognize and combine with specific antigen determinants.
One aspect of hybridoma technology concerns the fusion of myeloma cells with lymphocytes from animals which have been immunized with a particular antigen. Hybridomas manufacture monoclonal antibodies that are specific for a single antigenic determinant. Monoclonal antibodies are beginning to replace conventional antisera in standard diagnostic kits for such procedures as the radioimmunoassay. Significant work is also being done to adapt hybrodoma technology for therapeutic purposes.
Some properties that flow from an ideal parent cell line are (1) high fusion frequency; (2) high cloning efficiency; (3) the ability to grow rapidly in a serum medium; (4) no secretion of myeloma immumoglobulin (Ig) (5) stable production of large amounts of Ig after fusion; and (6) ability to grow when reinserted into the originating species. Immunoglobulin is the generic name for various isotypes of antibodies that include IgG, IgM, IgA, IgE, and the like. The various species of Ig have similarities and differences.
For example, all immunoglobulin molecules have a constant portion, that is highly conserved (i.e., constant) in amino acid sequence within a particular Ig subclass (e.g., IgG.sub.1). This constant region is responsible for various biological effector functions (e.g., complement activation). The portion of the immunoglobulin molecule responsible for immunological specificity (i.e., specific antigen binding) is called the variable region. It is made up of the variable regions of the Ig heavy and light chains. These variable regions differ in amino acid sequence according to the antigenic determinant which the Ig recognizes.
A typical procedure for making hybridomas is as follows: (a) immunize mice with a certain immunogen; (b) remove the spleens from the immunized mice and make a spleen suspension in an appropriate medium; (c) fuse the suspended spleen cells with mouse myeloma cells from a suitable cell line; (d) dilute and cultue in separate containers the mixture of unfused spleen cells, unfused myeloma cells and fused cells in a selective medium which will not support the unfused myeloma cells, for a time sufficient to allow death of all the unfused cells; (e) evaluate the supernatant in each container containing a hybridoma for the presence of antibody to the immunogen; and (f) select and clone hybridomas producing the desired antibodies. Once the desired hybridoma has been selected and cloned, the resultant antibody is produced by in vitro culturing of the desired hybrodoma in a suitable medium. In an alternative method, the desired hybridoma can be injected directly into mice intraperitoneally to produce an ascites.